NAFLD Project Summary/Abstract This research will utilize samples and data collected by the Nonalcoholic Steatohepatitis Clinical Research Network (NASH CRN) to develop a rapid, non-invasive and inexpensive diagnostic assay to detect and possibly stage non-alcoholic fatty liver disease (NAFLD). NAFLD is a progressive disease in which simple steatosis can lead to steatohepatitis, cirrhosis, liver failure and hepatocellular carcinoma. The prevalence of NAFLD is closely linked to obesity and obesity-related co-morbidities such as insulin resistance, diabetes, and dyslipidemia. Currently, liver biopsies are required to identify NAFLD stages of progression for patient diagnosis. This has greatly hindered NAFLD research because biopsy-related expenses and the risk of medical complications make the widespread screening of at-risk populations impractical. The proposed diagnostic will utilize a suite of fluorescently labeled fatty acid binding proteins to detect serum levels of hydrophobic metabolites, especially free fatty acids. By using multiple probes with distinct binding and signaling specificities, a probe-response profile that is characteristic of the patient's metabolic state is obtained. This novel, probe-based approach measures the unbound concentration of free fatty acids (FFA) in serum rather than the total concentration, which includes FFA bound to cell membranes and carrier proteins. Measurements of unbound FFA are preferable because they reveal how much of the FFA is readily available for receptor binding and other interactions or reactions. The profile of unbound FFA concentrations is distinct from the profile for total FFA, because different FFA reach different bound/unbound equilibriums with cell membranes and carrier proteins. In preliminary study utilizing 138 samples from the NASH-CRN and a suite of 12 probes with distinct fatty acid binding specificities we have shown that probe-response profiles can provide excellent separation of NAFLD patients from diabetics and healthy controls and provide discrimination between simple steatosis and severe NASH. The proposed studies will utilize high throughput methods for the profiling of serum samples and for the improvement of the diagnostic assay through the production and characterization of new probes. Specifically, the NASH-CRN repository of biopsy verified NAFLD patients and samples from other sources will be used to 1) verify that a suite of probes for serum metabolites can distinguish between NAFLD and non-NAFLD patients and controls, 2) develop and optimize a suite of probes for distinguishing between the medically recognized stages of NAFLD, 3) test whether profiling can be used to monitor the improvement of patients receiving therapy in NASH-CRN trials on adult (PIVENS) and pediatric (PIVENS) patients, and 4) complete the development of a suite of probes that can quantitatively measure a serum unbound FFA profile. Given the high prevalence of obesity in the United States, NAFLD is expected to create a substantial socioeconomic burden and it is critical that a noninvasive diagnostic for monitoring NASH in the general population be developed. Narrative Non-alcoholic fatty liver disease (NAFLD), which can lead to cirrhosis and liver cancer, is closely linked to obesity and is expected to affect 15% to 30% of the general population of the United States, including children. Because the only means of identifying the severity of NAFLD is a liver biopsy, which involves increased cost and the risk of medical complications, many at risk patients are not diagnosed and it is difficult to study the severity of the disease in the general population. The proposed research project will develop a rapid, non- invasive and inexpensive diagnostic assay to detect the presence and severity of NAFLD using only a drop of blood.